CD73 activity in cells

The CD73 activity assay was performed as described by Sherene Loi et al. [31]. MDA-MB-231 or 4 T1 was plated at 10⁴ cells per well in a 96-well plate and cultured in complete medium. After 24 h, culture medium was removed and cells were washed twice with prewarmed phosphate-free assay buffer (2 mM MgCl2, 125 mM NaCl, 1 mM KCl, 10 mM glucose, 10 mM Hepes pH 7.2, diluted in ddH2O). THF (12.5 or 25 μg/mL) diluted in assay buffer or buffer alone (control) was added to cells and incubated at 37 °C for 10 min. AMP (250 μM) diluted in assay buffer or buffer alone was then added and incubated at 37 °C for 1 min. Phosphate concentrations resulting from AMP hydrolysis by CD73 were measured using the malachite green phosphate detection kit (R&D Systems, MN, USA) following the manufacturer’s instructions. For further confirmation, THF, as well as APCP or tylosin (25 μg/mL), was tested again in this assay.